This invention relates to the field of gel electrophoresis, and in particular to cassette holders for slab-shaped electrophoresis gels and curved-surface cassette/gel systems. The invention may be incorporated into the multiple parallel slab gel electrophoresis system of U.S. Pat. No. 4,088,561, which is incorporated herein by reference.
Electrophoresis is a technique for separating molecules by virtue of their differential mobility in liquid medium under the influence of an electric field. Electrophoresis is usually performed in a gel medium to prevent convection in the fluid phase and the consequent loss of order and resolution. The gels used include polyacrylamide, agarose, and a variety of analogous substances.
Separations may be performed in gel rods, for individual samples, or in gel slabs, for a series of samples to be compared side-by-side or for two-dimensional separations. Typical dimensions of a gel slab for use in protein electrophoretic separation in the presence of sodium dodecyl sulfate (SDS) are 18 cm.times.18 cm.times.0.15 cm.
In many apparatus, including SDS-protein electrophoresis, the gel must be insulated on all surfaces except the two ends. A voltage is applied across these ends by means of contact between the gel ends and two buffer chambers containing electrodes, the anode in one chamber and the cathode in another. Such a configuration is usually implemented by surrounding the gel slab with an insulating cassette consisting of two sheets of insulator and insulating side spacers, forming a rectangular "tube" with a section open at both ends. The sheets are typically made of glass, plastic or aluminum oxide, and the spacers of glass, polyvinyl chloride or other plastic. The sheets cover the major surfaces of the gel. A major surface of a gel or cassette is the surface having the largest surface area.
Typically the gel is formed from liquid reagents poured into the cassette in order to achieve close contact between the gel thus created and the walls of the cassette. If the gel is formed by polymerization, then the gel will be in contact with all of the exposed internal surfaces of the cassette as is the case with acrylamide gels. This contact is important because any gaps between the gel and the cassette walls cause defects in the electrophoresis migration. In the case of the SDS-protein electrophoresis procedure, a liquid-filled space offers a localized region of increased conductivity, thereby destroying the symmetry of the electric field necessary to obtain informative patterns of separated proteins. A gas-filled gap gives rise to localized heating, due to the diminished thermal conductivity between the underlying gel and the external wall of the cassette. Due to power dissipated in the gel during passage of the electrophoretic current, good and even thermal conduction from the gel to the cassette walls and then to the surrounding cooling system is highly desirable.
The need to handle slab gels in the cassettes in which they were originally formed leads to a serious problem in that any transportation of gels between the sites of making and use must involve the transportation of the cassette, usually made of glass, which is fragile and heavy. In addition, this may necessitate the use of disposable cassettes in order to avoid the return shipment and cleanup of used cassettes. However, centralized manufacture of slab gels is very desirable as a means of providing a standardized separating medium to dispersed users of electrophoresis separations, and as a means of eliminating the need to produce the gels at the site of use, which is labor intensive.
Sufficiently close contact between gel and cassette is generally not achieved when a preformed slab gel is pressed between two flat sheets of stiff insulator, such as glass, because the pressure applied is generally localized and gaps, usually thin films of liquid, remain between glass and gel at other points. Such films disrupt the electrophoresis and give rise to imperfect separation patterns.
Recently, other investigators (P. Horowitz and S. Bowman, Electrophoresis. Vol. 7, pp. 534-535, 1986) have shown that a preformed gel attached by one surface to a flexible plastic backing can be successfully reassembled into a cassette having a pair of rigid glass or alumina walls which enclose the plane of the gel slab by "rolling the wet gel [having a GelBond backing] onto the front glass plate of the gel cassette." (GelBond is a trademark of FMC Corp., Rockland, Me.) The plastic backing effectively serves as one insulating wall of the cassette, and one that is flexible enough to allow any excess liquid to be squeezed from between the gel and the glass plate that forms the other insulating wall during application to the latter. The rolling process is generally accomplished by hand. The GelBond plastic produces no force normal to the plane of the gel slab to ensure that the seal formed during the rolling process is maintained. Therefore, it is not effective as a cassette wall to support a slab electrophoresis gel, and a second glass plate is required behind the GelBond. Clamping the cassette walls closed produces a non-uniform force normal to the surface of the gel slab so that the force is greater at the edges. This encourages the walls to bow outward forming a convex outer surface, allowing the formation of bubbles or free liquid droplets between the gel and plate at the middle of the gel area. The usefulness of the Horowitz system is therefore limited to gels of small size which can be enclosed in cassettes that are not subject to the bowing effect. Further, the plastic backing must be present in the Horowitz system because the gel can only be rolled onto a single rigid surface. This technique is difficult, is progressively more unwieldy for larger gels, and, most importantly, requires that the gel slab be on a plastic backing. Such a backing is expensive, makes the gels more difficult to manufacture, and prevents certain subsequent uses of the gels, such as so-called Western blotting.